Homozygous DBF4 mutation as a cause of severe congenital neutropenia.

BACKGROUND: Severe congenital neutropenia presents with recurrent infections early in life as a result of arrested granulopoiesis. Multiple genetic defects are known to block granulocyte differentiation; however, a genetic cause remains unknown in approximately 40% of cases. OBJECTIVE: We aimed to characterize a patient with severe congenital neutropenia and syndromic features without a genetic diagnosis. METHODS: Whole exome sequencing results were validated using flow cytometry, Western blotting, coimmunoprecipitation, quantitative PCR, cell cycle and proliferation analysis of lymphocytes and fibroblasts and granulocytic differentiation of primary CD34+ and HL-60 cells. RESULTS: We identified a homozygous missense mutation in DBF4 in a patient with mild extra-uterine growth retardation, facial dysmorphism and severe congenital neutropenia. DBF4 is the regulatory subunit of the CDC7 kinase, together known as DBF4-dependent kinase (DDK), the complex essential for DNA replication initiation. The DBF4 variant demonstrated impaired ability to bind CDC7, resulting in decreased DDK-mediated phosphorylation, defective S-phase entry and progression and impaired differentiation of granulocytes associated with activation of the p53-p21 pathway. The introduction of wild-type DBF4 into patient CD34+ cells rescued the promyelocyte differentiation arrest. CONCLUSION: Hypomorphic DBF4 mutation causes autosomal-recessive severe congenital neutropenia with syndromic features.

Disrupted Ca2+ homeostasis and immunodeficiency in patients with functional IP3 receptor subtype 3 defects.

Calcium signaling is essential for lymphocyte activation, with genetic disruptions of store-operated calcium (Ca2+) entry resulting in severe immunodeficiency. The inositol 1,4,5-trisphosphate receptor (IP3R), a homo- or heterotetramer of the IP3R1-3 isoforms, amplifies lymphocyte signaling by releasing Ca2+ from endoplasmic reticulum stores following antigen stimulation. Although knockout of all IP3R isoforms in mice causes immunodeficiency, the seeming redundancy of the isoforms is thought to explain the absence of variants in human immunodeficiency. In this study, we identified compound heterozygous variants of ITPR3 (a gene encoding IP3R subtype 3) in two unrelated Caucasian patients presenting with immunodeficiency. To determine whether ITPR3 variants act in a nonredundant manner and disrupt human immune responses, we characterized the Ca2+ signaling capacity, the lymphocyte response, and the clinical phenotype of these patients. We observed disrupted Ca2+ signaling in patient-derived fibroblasts and immune cells, with abnormal proliferation and activation responses following T-cell receptor stimulation. Reconstitution of IP3R3 in IP3R knockout cell lines led to the identification of variants as functional hypomorphs that showed reduced ability to discriminate between homeostatic and induced states, validating a genotype-phenotype link. These results demonstrate a functional link between defective endoplasmic reticulum Ca2+ channels and immunodeficiency and identify IP3Rs as diagnostic targets for patients with specific inborn errors of immunity. These results also extend the known cause of Ca2+-associated immunodeficiency from store-operated entry to impaired Ca2+ mobilization from the endoplasmic reticulum, revealing a broad sensitivity of lymphocytes to genetic defects in Ca2+ signaling.

Defective Sec61α1 underlies a novel cause of autosomal dominant severe congenital neutropenia.

BACKGROUND: The molecular cause of severe congenital neutropenia (SCN) is unknown in 30% to 50% of patients. SEC61A1 encodes the α-subunit of the Sec61 complex, which governs endoplasmic reticulum protein transport and passive calcium leakage. Recently, mutations in SEC61A1 were reported to be pathogenic in common variable immunodeficiency and glomerulocystic kidney disease. OBJECTIVE: Our aim was to expand the spectrum of SEC61A1-mediated disease to include autosomal dominant SCN. METHODS: Whole exome sequencing findings were validated, and reported mutations were compared by Western blotting, Ca2+ flux assays, differentiation of transduced HL-60 cells, in vitro differentiation of primary CD34 cells, quantitative PCR for unfolded protein response (UPR) genes, and single-cell RNA sequencing on whole bone marrow. RESULTS: We identified a novel de novo missense mutation in SEC61A1 (c.A275G;p.Q92R) in a patient with SCN who was born to nonconsanguineous Belgian parents. The mutation results in diminished protein expression, disturbed protein translocation, and an increase in calcium leakage from the endoplasmic reticulum. In vitro differentiation of CD34+ cells recapitulated the patient's clinical arrest in granulopoiesis. The impact of Q92R-Sec61α1 on neutrophil maturation was validated by using HL-60 cells, in which transduction reduced differentiation into CD11b+CD16+ cells. A potential mechanism for this defect is the uncontrolled initiation of the unfolded protein stress response, with single-cell analysis of primary bone marrow revealing perturbed UPR in myeloid precursors and in vitro differentiation of primary CD34+ cells revealing upregulation of CCAAT/enhancer-binding protein homologous protein and immunoglobulin heavy chain binding protein UPR-response genes. CONCLUSION: Specific mutations in SEC61A1 cause SCN through dysregulation of the UPR.

Murine myeloproliferative disorder as a consequence of impaired collaboration between dendritic cells and CD4 T cells.

Dendritic cells (DCs) are a key cell type in the initiation of the adaptive immune response. Recently, an additional role for DCs in suppressing myeloproliferation was discovered. Myeloproliferative disorder (MPD) was observed in murine studies with constitutive depletion of DCs, as well as in patients with congenital deficiency in DCs caused by mutations in GATA2 or IRF8 The mechanistic link between DC deficiency and MPD was not predicted through the known biology and has remained an enigma. Prevailing models suggest numerical DC deficiency leads to MPD through compensatory myeloid differentiation. Here, we formally tested whether MPD can also arise through a loss of DC function without numerical deficiency. Using mice whose DCs are deficient in antigen presentation, we find spontaneous MPD that is characterized by splenomegaly, neutrophilia, and extramedullary hematopoiesis, despite normal numbers of DCs. Disease development was dependent on loss of the MHC class II (MHCII) antigen-presenting complex on DCs and was eliminated in mice deficient in total lymphocytes. Mice lacking MHCII and CD4 T cells did not develop disease. Thus, MPD was paradoxically contingent on the presence of CD4 T cells and on a failure of DCs to activate CD4 T cells, trapping the cells in a naive Flt3 ligand-expressing state. These results identify a novel requirement for intercellular collaboration between DCs and CD4 T cells to regulate myeloid differentiation. Our findings support a new conceptual framework of DC biology in preventing MPD in mice and humans.

PD-L1 up-regulation restrains Th17 cell differentiation in STAT3 loss- and STAT1 gain-of-function patients.

Patients with hypomorphic mutations in STAT3 and patients with hypermorphic mutations in STAT1 share several clinical and cellular phenotypes suggesting overlapping pathophysiologic mechanisms. We, therefore, examined cytokine signaling and CD4+ T cell differentiation in these cohorts to characterize common pathways. As expected, differentiation of Th17 cells was impaired in both cohorts. We found that STAT1 was hyperphosphorylated in response to cytokine stimulation in both cohorts and that STAT1-dependent PD-L1 up-regulation-known to inhibit Th17 differentiation in mouse models-was markedly enhanced as well. Overexpression of SOCS3 strongly inhibited phosphorylation of STAT1 and PD-L1 up-regulation, suggesting that diminished SOCS3 expression may lead to the observed effects. Defects in Th17 differentiation could be partially overcome in vitro via PD-L1 inhibition and in a mouse model of STAT3 loss-of-function by crossing them with PD-1 knockout mice. PD-L1 may be a potential therapeutic target in several genetic diseases of immune deficiency affecting cytokine signaling.

Early-onset stroke and vasculopathy associated with mutations in ADA2.

BACKGROUND: We observed a syndrome of intermittent fevers, early-onset lacunar strokes and other neurovascular manifestations, livedoid rash, hepatosplenomegaly, and systemic vasculopathy in three unrelated patients. We suspected a genetic cause because the disorder presented in early childhood. METHODS: We performed whole-exome sequencing in the initial three patients and their unaffected parents and candidate-gene sequencing in three patients with a similar phenotype, as well as two young siblings with polyarteritis nodosa and one patient with small-vessel vasculitis. Enzyme assays, immunoblotting, immunohistochemical testing, flow cytometry, and cytokine profiling were performed on samples from the patients. To study protein function, we used morpholino-mediated knockdowns in zebrafish and short hairpin RNA knockdowns in U937 cells cultured with human dermal endothelial cells. RESULTS: All nine patients carried recessively inherited mutations in CECR1 (cat eye syndrome chromosome region, candidate 1), encoding adenosine deaminase 2 (ADA2), that were predicted to be deleterious; these mutations were rare or absent in healthy controls. Six patients were compound heterozygous for eight CECR1 mutations, whereas the three patients with polyarteritis nodosa or small-vessel vasculitis were homozygous for the p.Gly47Arg mutation. Patients had a marked reduction in the levels of ADA2 and ADA2-specific enzyme activity in the blood. Skin, liver, and brain biopsies revealed vasculopathic changes characterized by compromised endothelial integrity, endothelial cellular activation, and inflammation. Knockdown of a zebrafish ADA2 homologue caused intracranial hemorrhages and neutropenia - phenotypes that were prevented by coinjection with nonmutated (but not with mutated) human CECR1. Monocytes from patients induced damage in cocultured endothelial-cell layers. CONCLUSIONS: Loss-of-function mutations in CECR1 were associated with a spectrum of vascular and inflammatory phenotypes, ranging from early-onset recurrent stroke to systemic vasculopathy or vasculitis. (Funded by the National Institutes of Health Intramural Research Programs and others.).

Diminished allergic disease in patients with STAT3 mutations reveals a role for STAT3 signaling in mast cell degranulation.

BACKGROUND: Severe atopic conditions associated with elevated serum IgE are heterogeneous with few known causes. Nearly every patient with autosomal-dominant hyper-IgE syndrome (AD-HIES) due to signal transducer and activator of transcription 3 (STAT3) mutations has a history of eczematous dermatitis and elevated IgE; however, clinical atopy has never been systematically studied. OBJECTIVE: Understanding of genetic determinants of allergic disease may lead to novel therapies in controlling allergic disease. METHODS: We conducted clinical evaluation of the rates of food allergies and anaphylaxis in patients with AD-HIES, a cohort of patients with no STAT3 mutation but with similar histories of elevated IgE and atopic dermatitis, and healthy volunteers with no history of atopy. Morphine skin prick testing, ImmunoCAP assays for allergen-specific IgE, and basophil activation were measured. A model of systemic anaphylaxis was studied in transgenic mice carrying an AD-HIES mutation. STAT3 was silenced in LAD2 and primary human mast cells to study the role of STAT3 in signaling and degranulation after IgE cross-linking. RESULTS: Food allergies and anaphylaxis were markedly diminished in patients with AD-HIES compared with a cohort of patients with no STAT3 mutation but with similar histories of elevated IgE and atopic dermatitis. Morphine skin prick testing and basophil activation were diminished in patients with AD-HIES, whereas mice carrying an AD-HIES mutation were hyporesponsive to systemic anaphylaxis models. Rapid mast cell STAT3 serine727 phosphorylation was noted after IgE cross-linking, and inhibition of STAT3 signaling in mast cells lead to impaired FcεRI-mediated proximal and distal signaling, as well as reduced degranulation. CONCLUSION: This study serves as an example for how mutations in specific atopic pathways can lead to discrete allergic phenotypes, encompassing increased risk of some phenotypes but a relative protection from others.

Peptide library-based evaluation of T-cell receptor breadth detects defects in global and regulatory activation in human immunologic diseases.

The ability of T-cells to respond to foreign antigens and to appropriately regulate this response is crucial for maintaining immune homeostasis. Using combinatorial peptide libraries, we functionally measured broad T-cell reactivity and observed impaired reactivity in established models of T-cell receptor repertoire restriction and in previously unrecognized disease contexts. By concurrently analyzing T-regulatory and T-effector cells, we show strong functional correlation between these subsets in healthy individuals and, strikingly, that alterations of this balance are associated with T helper type 2 (Th2)-mediated disease in a lymphopenic setting. Finally, we demonstrate that peptide-based priming of polyclonal naive cells with relatively low concentrations skews toward Th2 differentiation. These findings provide unique insight into the pathophysiology and functional consequences of abnormal T-cell repertoires and into differentiation of human naive T-cells.